[Regulatory effect of Egr-1 promoter sequences induced by doxorubicin in transcriptional targeting on expression of GM-CSF gene].
In order to explore the regulatory effects of Egr-1 promoter sequences induced by doxorubicin (ADM) in transcriptional targeting on the expression of hematopoietic growth factor genes. The human GM-CSF cDNA and enhanced green fluorescent protein (eGFP) cDNA were linked together with internal ribozyme entry site (IRES) and then inserted into the expression vector pCIneo under control of the Egr-1 promoter (Egr-EG). The vector was transferred into human bone marrow stromal cell line HFCL by lipofectin. The transfected cell clones (HFCL/EG) were selected by the addition of G418. The cells were exposed to the clinically important anticancer agent doxorubicin. The activity of eGFP in HFCL/EG cells was detected by flow cytometry. The amounts of GM-CSF in HFCL/EG postchemotherapy were confirmed with ELISA. The effect of GM-CSF in HFCL/EG cultural supernatants on expansion of CFU-GM derived from cord blood was also studied. The effect of N-acetylcysteine (a free radical scavenger) on GM-CSF production following exposure to ADM was examined. The results indicated that the activity of eGFP and the amounts of secreted GM-CSF in HFCL/EG cells exposed to ADM increased as compared to non-ADM group. The effect of GM-CSF in HFCL/EG cultural supernatants on expansion of CFU-GM was significantly higher than that of non-ADM group. N-acetylcysteine significantly decreased the concentration of GM-CSF produced by HFCL/EG treated with ADM. It is concluded that these in vitro data provide an experimental basis for the use of gene therapy of hematopoietic growth factor gene regulated by Egr-1 promoter to protect hematopoiesis from ADM-injury.
DU
Nan
N
Department of Oncology, The First Hospital, PLA General Hospital, Beijing 100037, China. dunan05@yahoo.com.cn
Pei
Xue-Tao
XT
Sun
Jun-Zhong
JZ
Fu
Yan
Y
Zhao
Hui
H
Wang
Xi-Liang
XL
D001483:Base Sequence; D001854:Bone Marrow Cells / Q000166:cytology; D002460:Cell Line; D004317:Doxorubicin / Q000494:pharmacology*; D051766:Early Growth Response Protein 1 / Q000235:genetics*; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics*; D006801:Humans; D011401:Promoter Regions, Genetic
C497325:EGR1 protein, human; D051766:Early Growth Response Protein 1; D004317:Doxorubicin; D016178:Granulocyte-Macrophage Colony-Stimulating Factor